IgA is the predominant immunoglobulin molecule synthesized in the human body. Most of this IgA is produced by plasma cells residing in the intestinal lamina propria and is transported across the epithelium and into the lumen as secretory IgA that helps to establish and maintain homeostasis with the commensal bacteria that stably reside in the human gastrointestinal tract. Intestinal Peyer's patches are important anatomic sites for the induction of IgA responses. In order to stimulate the production of IgA, commensal bacteria and bacterial antigens must first be taken up by antigen-sampling cells. There are two antigen-sampling pathways that are candidates to explain how bacteria and bacterial antigens are initially taken up into Peyer's patches: microfold (M) cell-mediated uptake and direct sampling of luminal bacteria by mononuclear phagocytes (including both dendritic cells and macrophages). M cells are specialized antigen-sampling epithelial cells that are found in the follicle-associated epithelium (FAE) covering organized lymphoid structures in the small intestine and colon including Peyer's patches and isolated lymphoid follicles. The goal of this project is to use a mouse model system to determine if M cells play a dominant role compared to other potential antigen-sampling pathways in the initiation of secretory IgA production. The cytokine RANKL is necessary and sufficient to initiate M cell differentiation from uncommitted epithelial precursor cells in intestinal crypts. As a result, mice with a conditional deletion of RANK in the intestinal epithelium (RANK IEC mice) lack intestinal M cells in their Peyer's patches. Preliminary studies show that conventionally housed RANK IEC mice have significant deficiencies in the amount of fecal IgA produced and the density of IgA+ plasma cells present in the intestinal lamina propria. This project will use germ-free mice and mice recolonized with a defined set of anaerobic enteric bacteria (Altered Schaedler Flora or ASF) to determine whether secretory IgA production to this defined set of bacteria is also dependent on bacterial uptake by M cells. The central hypothesis guiding the proposed experiments is that M cell-mediated antigen sampling accounts for most sampling of commensal bacteria at the site of inductive intestinal lymphoid tissues, thereby initiating the efficient induction of the normal secretory IgA response to bacterial antigens. The first aim of the proposal is to determine the impact of absence of intestinal M cells on secretory IgA production by germ-free mice. The second aim is to characterize the secretory IgA response to introduction of a defined set of commensal microbiota (ASF) into M cell-deficient RANK IEC mice and control littermates. Mechanistic insights into how M cell-mediated antigen sampling by the gut immune system promotes intestinal homeostasis may be useful in developing improved oral vaccination strategies and new approaches to the treatment of human inflammatory bowel disease.